Crustacean expression vector

ABSTRACT

Methods and constructs for genetic manipulation of one or more of shrimp, shellfish, mollusks, and fish are disclosed. The nucleic acid construct includes a promoter and an internal ribosome entry site of an insect picomavirus, such as a cricket paralysis-like picomavirus. One or more open reading frames can be operably associated with one or both of the promoter and the internal ribosome entry site, and one or more proteins or protein subunits can be expressed upon introduction of the construct into a host cell, such as into a shrimp. Method for producing immortalized crustacean cell lines using enhancer elements derived from shrimp and/or shrimp viruses are also described.

BACKGROUND OF THE INVENTION

Fish and shellfish farming significantly contribute to the global foodsupply and is a source of a major economic activity in developingnations (FAO Fisheries department, 2000). As the supply of food fish andshellfish from capture fisheries declines globally, there is an urgentneed to enhance aquaculture production. The development of aquaculturein a sustainable manner faces a number of challenges. Among themdiseases caused by diverse etiologic agents is of particular importance.Disease outbreaks and emergence of new pathogens poses a major challengefor sustainable development in aquaculture. A case in point is shrimpaquaculture.

During the past decade, shrimp (Penaeus sp.) farming has evolved fromsubsistence level farming to a major worldwide industry providing jobsto millions of people both directly and indirectly, particularly in thecoastal areas of the developing nations in the Asia and the Central andSouth Americas. Among the challenges facing shrimp farming globally,economic losses from diseases caused by viruses are a major concern.Since the first report of a viral disease in early 1970s, more than 20viruses have been reported that infect shrimp (Lightner, 1996, Rev. Sci.Tech. Off. Int. Epiz. 15:579-601). This list is growing rapidly. Many ofshrimp viruses have caused serious epizootics in penaeid shrimpresulting in significant economic losses to commercial shrimp farmersand potentially affecting wild crustacean populations adversely. Thefour most important viruses of penaeid shrimp are white spot syndromevirus (WSSV), yellowhead virus (YHV), Taura syndrome virus (TSV) and theinfectious hypodermal and hematopoietic necrosis virus (IHHNV). WSSV,YHV, and, more recently, TSV have caused serious epizootics in theEastern Hemisphere; whereas, WSSV, TSV and IHHNV related economic losseshave occurred in the Western Hemisphere (Dhar et al., 2004, Adv. VirusRes., In press; Tu et al., 1999, Dis. Aquat. Org. 38:159-161; Lightneret al., 1996, Rev. Sci. Tech. Off. Int. Epiz. 16:146-160). Considerableprogress has been made in developing detection methods andcharacterizing these viral pathogens at molecular level over the lastfew years. However, information on the role of the virally encodedproteins in viral pathogenesis and the genes involved in host anti-viralresponse remains largely unknown. This is primarily because of: (1) alack of a suitable transient and transfection vectors for shrimp andother crustaceans and (2) a lack of a permanent cell line for shrimp andany other crustaceans.

The instant invention addresses these issues by developing expressionvectors for transient expression of foreign genes, and for transfectionof shrimp primary cell lines with foreign genes or modifiers ofendogenous genes. These vectors could be used (1) to express recombinantprotein(s) with therapeutic potential using shrimp or other crustaceanhost, (2) to express host gene or foreign gene in excess to determinetheir role in growth, development, and or disease resistance usingshrimp or other crustacean host, (3) to develop a transgenic shrimp orother crustaceans, and (4) to study the role of virally encoded proteinin viral pathogenesis in vitro and in vivo. These techniques willdevelop the tools needed to modify primary cell cultures to enable thedevelopment of unregulated growth necessary for immortalized cell lines.

SUMMARY OF THE INVENTION

The inventors have discovered a number of important tools that willenable the manipulation of animal genes both transiently and permanentlyin both tissue culture and live animals. In general, the inventionharnesses elements from shrimp viruses that through bioinformaticsevaluation have been shown to have homology to enhancer or regulatoryelements in other species. The inventors have demonstrated specificvectors that can be developed from these elements and methods of theiruse in the instant invention that will allow the improvement of animalhealth as well as diagnostic uses thereof. Specifically the inventionrelates to improvement of crustacean and especially shrimp health inrelation to virally transmitted disease. The invention also providestools for drug development and diagnostics of the same.

It is an object of the invention to provide a transient expressionvector for the expression of a foreign gene or genes in Penaeid shrimptissues in vitro or in vivo.

It is a further object of the invention to provide a transientexpression vector for the expression of a foreign gene or genes inbacteria and yeast.

It is an object of the invention to provide a transfection vector forthe expression of a foreign gene or genes in Penaeid shrimp tissue invitro or in vivo.

It is an object of the invention to provide a transfection vector fordeveloping transgenic shrimp expressing a foreign gene or shrimp gene orany combination thereof.

It is an object of the invention to provide a transient and atransfection vector for developing a permanent cell line for shrimp,crustaceans, and mollusks by over expressing host telomerase gene orintroduction of a heterologous telomerase gene.

It is an object of the invention to provide a transfection vector fordeveloping transgenic fish, shellfish or insects expressing a foreigngene or genes. Such a vector would have a broad host range that may beapplicable to other animals as an expression system.

It is an object of the invention to provide methods of transientexpression in crustaceans, fish, shellfish, insects, or other cellculture based on the vectors developed herein.

It is an object of the invention to provide methods for transfection incrustaceans, fish, shellfish, insects, or other cell culture based onthe vectors developed herein.

It is an object of the invention to provide methods of protecting shrimpfrom WSSV, IHHNV, TSV, YHV and other viral, bacterial and fungaldiseases by over expressing antiviral/antimicrobial factors using thetransfection vector.

It is an object of the invention to provide methods of protecting shrimpfrom WSSV, IHHNV, TSV, YHV and other viral, bacteria, and fungaldiseases by over expressing pathogen-encoded protein (e.g., capsidprotein) using the transfection vector.

It is an object of the invention to provide methods of protecting fishand shellfish from viral, bacterial, fungal and or other microbialinfection by over-expressing antiviral/antimicrobial factors using thetransfection vector for delivery of the therapeutic effect.

It is an object of the invention to provide methods of protecting fishand shellfish from viral, bacterial, fungal and or other microbialinfection by over expressing pathogen-encoded protein or proteins (e.g.,capsid protein, enzyme, receptor, recognition sequence) using thetransfection vector.

Therefore, the invention provides a composition for developing atransient or transfection vector using sequences of shrimp virus andshrimp cellular gene as well as the methods for using such vectors formanipulation of cells and diagnostic applications. Such vectors could beused for the expression of recombinant protein in vitro or in vivo inshrimp or other crustaceans, fish, insects, and other animals.

BRIEF DESCRIPTIONS OF THE FIGURES

FIG. 1 consists of FIGS. 1A, 1B, 1C, and 1D. A comparativerepresentation of the genomic organization of several mammalian andinsect picomaviruses and plant RNA viruses—PV in FIG. 1A, IFV in FIG.1B, TSV in FIG. 1C, and PYFV in FIG. 1D. Abbreviations used are asfollows: ORF=Open reading frame; UTR=untranslated region; VPg=genomelinked protein; and ?=the presence of VPg has not been confirmed. Thehelicase (triangle), protease (circle), and the RNA dependent RNApolymerase (square) regions are indicated.

FIG. 2 consists of FIGS. 2A, 2B, and 2C. In these figures, theintergenic regions between ORF1 and ORF 2 of TSV are aligned andcompared with the homologous regions of insect picoma-like viruses. Thesequences in FIG. 2A continue in FIG. 2B, and those in FIG. 2B continuein FIG. 2C. Nucleotide residues indicated in bold, underlined text areconserved residues, underlined (but not bold) residues are partiallyconserved among the sequences, and residues indicated in ordinary textappear not to be conserved. The numbers presents the correspondingnumber of the first nucleotide in the intergenic region between ORF1 andORF2, except that in order to improve the alignment, the first 350nucleotide residues of RhPV are not shown. Corresponding SEQ ID NOs:are, for cricket paralysis viruse (CrPV), SEQ ID NO: 1; for Drosophila Cvirus (DCV), SEQ ID NO: 2; for Plautia stali intestine virus (PSIV), SEQID NO: 3; for Himetobi P virus (HiPV), SEQ ID NO: 4; for black queencell virus (BQCV), SEQ ID NO: 5; for triatoma virus (TrV), SEQ ID NO: 6;for Rhopalosiphum padi virus (RbPV), SEQ ID NO: 7; for Taura syndromevirus (TSV), SEQ ID NO: 8; for acute bee paralysis virus (ABPV), SEQ IDNO: 9.

FIG. 3 is a schematic representation of the genome organization ofIHHNV, based on the sequence of GenBank accession no. AF273215. Thenumbers on the diagram indicate nucleotide residue numbers. The leftopen reading frame (ORF) starts at nucleotide residue 313 and ends atresidue 2596, the middle ORF starts at nucleotide residue 534 and endsat residue 1631, and the right ORF starts at nucleotide residue 2535 andends at residue 3527. The IHHNV promoters, P2 and P61, are indicated byopen triangles.

FIG. 4 is an image of results of an agarose gel electrophoreses of theIHHNV P61 amplicon. PCR amplified DNA was electrophoresed in a 1.5%agarose gel containing ethidium bromide and imaged. An arrow indicatesthe 165 residue P61 amplicon. M represents a 100 base pair DNA ladderstandard. Lanes 1 through 4 represent DNA from 4 differentIHHNV-infected shrimp that was used for PCR.

FIG. 5 is a circular nucleotide map of the WSSV DNA genome(Thai-isolate, GenBank accession no. AF369029). The nucleotide residuedesignated 1 is the nucleotide A of ATG codon of the capsid protein geneVP28. The arrangement of all putative genes in both strands is shown byboxes on the ring inside the scale. Bars on the dashed line indicate thelocation of the repeat regions throughout the genome. The locations ofthe viral capsid genes (VP) are indicated by bars on the dotted line inthe map. The ring within the ring indicating viral capsid genesindicates the GC skew, and the ring in the center represents G skew.

FIG. 6 is a BLASTX search and amino acid alignment of WSSV transposaseamino acid sequence (SEQ ID NO: 10; WSSVT is the transposase ofWSSV-Taiwan isolate ORF 166) and a bacterial transposon amino acidsequence (SEQ ID NO: 11; SESET is a transposon of Salmonella entericasubsp. enterica serovar Typhi; GenBank Accession no. 16760800).

FIG. 7 is a schematic map of the transposase gene in WSSV isolate Taiwan(GenBank accession number AF440570). Inverted repeat sequences areindicated by the abbreviation IR. Viral repeat sequence (SEQ ID NO: 12)is indicated in the Repeat box. The numbers on top of the boxes (238587and 239925) indicate the nucleotide position in the genome of theWSSV-Taiwan isolate. The length 1337 bp indicates the length oftransposase gene, in base pairs. The palindromic sequence within theinverted repeat sequences (SEQ ID NOs: 13 and 14) is underlined.

FIG. 8 is a BLASTX search and amino acid sequence alignment of shrimpDNA clone AF077579 amino acid sequence (SEQ ID NO: 15; “Shrimp”; Xu etal., 1999, Animal Genet. 30:1-7) and the non-LTR Penelope transposableelement of Drosophila virilis amino acid sequence (SEQ ID NO: 16; fromGenBank Accession no. Q24736).

FIG. 9, consisting of FIGS. 9A and 9B, is a schematic representation ofshrimp expression vectors described herein. The vector diagramed in FIG.9A is a transient expression vector containing the IHHNV P61 promoter,IRES element of the TSV intergenic region between ORF1 and ORF2, and amarker gene. The vector diagramed in FIG. 9B is a transfection vectorfor shrimp containing the IHHNV P61 promoter, shrimp Penelopefull-length ORF, TSV IRES element and a selectable marker (e.g.,pDsRed). Shrimp Penelope represents the full-length copy of the Penelopegene from shrimp. Arrows marked by 5′ and 3′ represent the positions ofthe shrimp Penelope element inverted terminal repeats.

FIG. 10 is a schematic representation of a shrimp transfectionexpression vector described herein. A transfection vector for shrimp cancontain the IHHNV P61 promoter, full-length WSSV transposase gene, TSVIRES element and a selectable marker (e.g., pDsRed). Arrows marked by 5′and 3′ represent the positions of the WSSV inverted terminal repeats.

FIG. 11 is a map of a plasmid constructed by inserting the IHHNv p61promoter into the pGL3-Basic vector (Promega).

FIG. 12 is an image of an agarose gel separation of plasmids produced inExample 15 herein. Lane 1 contains a 1 kilobase DNA standard, Lanes 2-5contain linearized pGL3 enhancer vector, Lane 6 contains a 100 base pairDNA standard, Lanes 7 & 8 contain 25 bp DNA standard, and Lanes 8-11contain individual pSTF isolates double digested with SacI and NheI.

FIG. 13 is a map of a pSTH vector constructed using the IHHNV p61promoter and the pGL3-Enhancer vector as a backbone.

FIG. 14 is a map of a pSTI vector constructed using the IHHNV p2promoter and the pGL3-Enhancer vector as a backbone.

FIG. 15, consisting of FIGS. 15A and 15B, is a pair of plasmid maps ofIHHNV p61 promoter driving DsRed2 expression without added IRES element(PSTD, shown in FIG. 15A) and with the TSV IRES element (PSTE, shown inFIG. 15B).

FIG. 16 is an image of an agarose gel separation of the digest describedin Example 16 herein. Lane 1 contains a 100 base pair DNA ladderstandard. Lane 2-TSV contains the IRES PCR product.

FIG. 17 is a standard curve generated for the luciferase assay used todetermine expression of luciferase in shrimp tissues, as described inExample 17 herein.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to compositions and methods that can be used toexpress heterologous nucleic acids and proteins in cells of marinecreatures, including crustaceans such as shrimp, lobster, crayfish, andcrabs, and further including non-crustacean arthropods and other marineanimals such as fish and mollusks.

The invention relates to a non-naturally-occurring nucleic acidconstruct that includes at least a viral promoter region and an insectpicomavirus (IPV) internal ribosome entry site (IRES). One or both ofthe promoter and the IRES can be linked with one or more cistrons, suchas an intron-free open reading frame (ORF). Significantly, it has beendiscovered that use of a virus promoter and an IRES from an IPV rendersthe construct capable of expressing in a crustacean cell RNA or proteinencoded by a nucleic acid introduced into the cell as part of theconstruct. Such constructs can be used to produce one or more nucleicacids in a host crustacean cell. By way of example, an mRNA encoding aprotein of interest can be produced, as can an RNA that binds with aspecific endogenously-produced nucleic acid of the cell. Further by wayof example, a protein encoded by a portion of the construct can producedin a crustacean cell by incorporating into the cell a construct in whichthe portion is operably linked to a viral promoter to generate RNA thatincludes both the portion and an IPV IRES, which can be translated bythe host cell to produce the protein.

The identities of the virus from which the promoter and the IPV fromwhich the IRES are derived is not critical. Preferably, the promoter andthe IRES are derived from the same IPV, but elements from distinctviruses can be used as well. The IPV(s) can be substantially any IPVthat is currently known or hereafter developed. A preferred type of IPVis the class of IPVs that are designated “cricket paralysis-like”picornaviruses, as described in the art (e.g., Christian et al., 1998,In: Miller et al., Eds., “The Insect Viruses” New York, Plenum Press,pp. 301-329; van Regenmortel et al., 2000, In: “Virus Taxonomy: TheClassification and Nomenclature of Viruses. The Seventh Report of theInternational Committee on Taxonomy of Viruses” Academic Press, SanDiego). It is recognized that it is not always possible to distinguishpicornaviruses from other RNA-containing viruses which exhibitsignificant morphological, physical, and chemical similarity topicornaviruses. Thus, the art recognizes a group of “picorna-like”viruses, such as TSV of shrimp. As used herein, the term IPV includesboth viruses that are capable of infecting hosts in phylum Arthropoda,regardless of whether they are designated “picornaviruses” or“picorna-like viruses.”

Knowledge of the identity of a host cell into which a nucleic acidconstruct of the type described herein will be incorporated can guideselection of an appropriate IPV. It has been discovered that promotersfrom substantially any virus (at least viruses suitable for any actualor anticipated host for the construct) and IRES' from substantially anyIPV can be used in vectors intended for use in crustaceans. Nonetheless,it can be preferable to select a promoter, an IRES, or both, from avirus that is known to infect crustaceans of the same species as thecrustacean from which the host cell was derived. Thus, for example, if anucleic acid construct described herein is to be used to produce aprotein upon incorporation into shrimp cells, it can be preferable touse a promoter and IRES from among those of known shrimp viruses, suchas infectious hypodermal and hematopoietic necrosis virus (IHHNv), Taurasyndrome virus (TSV), or white spot syndrome virus (WSSV).

The properties of many viral promoters are known, including multiple IPVpromoters. Substantially any viral promoter can be used in the nucleicacid constructs described herein. Examples of suitable promoters includepromoters from TSV, IHHNV, and WSSV (e.g., the P2 or P61 promoter ofWSSV).

In many picomaviruses, especially including those having genes arrangedin a single polycistronic unit, an IRES occurs in the 5′-untranslatedregion of the viral genome. In other picomaviruses, an IRES occurs inthe intergenic region between two or more cistronic regions of the viralgenome. Either type of IRES can be incorporated into the nucleic acidconstruct described herein.

The nucleic acid constructs described herein can be designed to expressa product for a relatively short time or for a longer period, such asthe lifetime of a host cell or organism, or even across generations oforganisms. The association between the form of a nucleic acid construct(e.g., straight-chain nucleic acid, plasmid, or viral vector) and itsduration in vivo are known and a skilled artisan can influence theduration of product production from the construct by selection of asuitable form of the construct.

Duration of product production can also be influenced by the propertiesof the genetic elements included in the construct. By way of example,the promoter that is used can be an inducible or repressible promoter,subject to regulation by its effectors. Similarly, it is known thatpromoters can induce transcription of an operably linked sequence at ahigh or low level, depending on the identity of the promoter and thehost organism.

Residence in a host cell of a construct described herein can beinfluenced by the presence or absence of genetic elements in theconstruct. By way of example, if the construct in introduced into a hostcell in the form of a plasmid, the plasmid (together with the construct)will be replicated if the plasmid includes an origin of replication site(ORS) that is operable in the host cell. It is known that ORS can inducemaintenance of single, a few, or multiple copies of a plasmid in a cell,and a skilled artisan can select an appropriate ORS for a desired copynumber for a desired host cell. Further by way of example, if theconstruct is included in a virus vector construct that is capable ofreplication and packaging (or can be induced to replicate and package),then maintenance or spread of the construct can be controlled usingmethods understood by a skilled artisan in this field.

One or more cistronic elements (e.g., an ORF) can be operably linkedwith the IRES of the construct, to thereby allow expression of theprotein or protein subunit encoded by the cistron. The cistronic elementshould also be operably linked with the promoter, so as to enhancetranscription of RNA encoding the cistron prior to translation broughtabout by introduction of a ribosome into the RNA by way of the IRES (orby a normal, mRNA cap-targeted ribosome or both). The cistron ispreferably an ORF that is not interrupted by an intron, but one or moreintrons can be present so long as the host cell is able to remove theintron(s) to yield the construct-encoded product of interest.

A cistron of the construct can encode an RNA that has a physiologicaleffect on the host cell (e.g., one that hybridizes specifically with anucleic acid in the cell). The cistron can also encode a protein. Forexample, if the cell is a cell of a crustacean food product (e.g., afarm-raised shrimp), the protein can be one which enhances the humannutritional value, the health of the crustacean, or the diseaseresistance of the crustacean. Proteins which enhance the lifespan,physiology or rate of proliferation of an individual cell are furtherexamples of proteins that can be expressed in a host cell from thenucleic acid construct described herein.

It is known that a protein or nucleic acid expressed in a cell can exerta direct physiological effect on a cell (e.g., a protein that catalyzesa chemical reaction or is a structural component of a cell or a nucleicacid that binds with a cellular nucleic acid in way that affectsexpression of the cellular nucleic acid). It is also known that aprotein or nucleic acid expressed in a cell can exert a relativelyindirect physiological effect on a cell (e.g., a protein that acts as atranscription factor or a nucleic acid that modulates the activity of acellular nucleic acid polymerase). It is immaterial which of these (orother) types of products is produced from a nucleic acid constructdescribed herein when the construct is introduced into a host cell. Askilled artisan can select an appropriate product based on the identityand biochemical properties of the host cell and the desired effect, andno more than ordinary experimentation is required to make a constructthat produces the appropriate product. Such products can be productsthat are ordinarily produced in the host cell (e.g., at a differentlevel in the presence and absence of the construct) or can beheterologous products (e.g., products unlike any produced by the hostcell or an altered form of a homologous product).

A transposable element (TE) can be included in the construct if desired.A TE facilitates distribution of operably linked elements of theconstruct into the genome of the host cell. A TE can thereby incorporatea transgene from the construct described herein (transiently orrelatively permanently, depending on the TE selected) into the hostgenome. A skilled artisan is able to operably link selected elements ofthe construct described herein with a TE with no more than routineexperimentation. Examples of suitable TEs include those that arehomologous to the Drosophila Penelope TE sequence reported in theliterature. Other suitable TEs include IPV TEs, such as those of WSSV.

In an important embodiment, the nucleic acid construct described hereincan be used to express a protein in a host cell, such as a crustaceancell. This is achieved by introducing into the host cell one or morecopies of the nucleic acid construct described herein. The method bywhich the construct is introduced into the host cell is not critical,and a variety of methods of introducing a nucleic acid vector into acell are known in the art. Examples of suitable methods of incorporatingthe construct into a host cell include use of a virus vector containingthe construct, a ballistically administered particle coated with orcontaining the construct, and electroporesis of cells in the presence ofa plasmid that includes the construct.

Definitions

In describing the present invention, the following terminology is usedin accordance with the definitions set out below.

“Shrimp” refers to any of the group of crustaceans that are commonlycultured for aquaculture or captured in the wild fisheries. The term“shrimp” includes shrimp eggs, shrimp larvae, shrimp post-larvae andadult shrimp. The term “shrimp” and “prawn” will be used interchangeablythroughout the specification. Shrimp can refer to but are not limitedto, Penaeus shrimp and include the species Penaeus vannamei, Penaeuschinensis, Penaeus monodon, Penaeus stylirostris, Penaeus japonicus,Penaeus penicillatus, Penaeus merguiensis, Penaeus indicus, Penaeussubtilis, Penaeus paulensis, Penaeus setiferus, Penaeus brasiliensis,Penaeus duorarum, Penaeus occidentalis, Penaeus schmitti, Penaeuscaliforniensis, Penaeus semisulcatus, Penaeus latisulcatus, Metapenaeusmonoceros, Metapenaeus dobsoni, Metapenaeus affinis, and Metapenaeusbrivicornis; and Litopenaeid shrimp (such as Litopenaeus vannamei, L.setiferus).

A “vector” comprises a nucleic acid, which can infect, transfect, andtransiently or permanently transform or transfect a cell. It will berecognized that a vector can be a naked nucleic acid, or a nucleic acidcomplexed with protein or lipid. The vector optionally comprises viralor bacterial nucleic acids and/or proteins, and/or membranes (e.g., acell membrane, a viral lipid envelope, etc.). Vectors include, but arenot limited to replicons (e.g., RNA replicons, bacteriophages) to whichfragments of DNA may be attached and become replicated. Vectors thusinclude, but are not limited to RNA, autonomous self-replicatingcircular or linear DNA or RNA (e.g., plasmids, viruses, and the like,see, e.g., U.S. Pat. No.5,217,879), and include both the expression andnon-expression plasmids. Where a recombinant microorganism or cellculture is described as hosting an “expression vector” this includesboth extra-chromosomal circular and linear DNA and DNA that has beenincorporated into the host chromosome(s). Where a vector is beingmaintained in a host cell, the vector may either be stably replicated bythe cells during mitosis as an autonomous structure, or is incorporatedwithin the host's genome.

The term “promoter” includes all sequences capable of drivingtranscription of a coding sequence in a cell, e.g., a plant cell, animalcell, bacterial cell, fungal cell, and yeast cell. Thus, promoters usedin the constructs of the invention include cis-acting transcriptionalcontrol elements and regulatory sequences that are involved inregulating or modulating the timing and/or rate of transcription of agene or genes. For example, a promoter can be a cis-actingtranscriptional control element, including an enhancer, a promoter, atranscription terminator, an origin of replication, a chromosomalintegration sequence, 5′ and 3′ untranslated regions, or an intronicsequence, which are involved in transcriptional regulation. Thesecis-acting sequences typically interact with proteins or otherbiomolecules to carry out (turn on/off, regulate, modulate, etc.)transcription. “Constitutive” promoters are those that drive expressioncontinuously under most environmental conditions and states ofdevelopment or cell differentiation. “Inducible” or “regulatable”promoters direct expression of the nucleic acid of the invention underthe influence of environmental conditions or developmental conditions.Examples of environmental conditions that may affect transcription byinducible promoters include anaerobic conditions, elevated temperature,drought, or the presence of light.

“Plasmids” can be commercially available, publicly available on anunrestricted basis, or can be constructed from available plasmids inaccord with published procedures. Equivalent plasmids to those describedherein are known in the art and will be apparent to the ordinarilyskilled artisan.

“Transposable elements” (TEs) or “transposons” are a group of geneticelements that move from one locus to another by non-homologousrecombination.

“Internal ribosome entry site” or “IRES” refers to a translation controlelement for cap independent mRNA translation. IRES' have been describedin the art (e.g., see review by Hellen et al., 2001, Genes Develop.15:1596-1612), and a skilled artisan is able to operably link an openreading frame or cistron with an IRES with no more than routineexperimentation.

The term “gene” includes a nucleic acid sequence comprising a segment ofDNA involved in producing a transcription product (e.g., a message),which in turn is translated to produce a polypeptide chain, or regulatesgene transcription, reproduction or stability. Genes can include regionspreceding and following the coding region, such as leader and trailer,promoters and enhancers, as well as, where applicable, interveningsequences (introns) between individual coding segments (exons).

The phrases “nucleic acid” or “nucleic acid sequence” includesoligonucleotide, nucleotide, polynucleotide, or to a fragment of any ofthese, to DNA or RNA (e.g., mRNA, rRNA, tRNA) of genomic or syntheticorigin which may be single-stranded (ss) or double-stranded (ds) and mayrepresent a sense (+) or antisense (−) strand, to peptide nucleic acid(PNA), or to any DNA-like or RNA-like material, natural or synthetic inorigin, including, e.g., iRNA, ribonucleoproteins (e.g., iRNPs). Theterm encompasses nucleic acids, i.e., oligonucleotides, containing knownanalogues of natural nucleotides. The term also encompassesnucleic-acid-like structures with synthetic backbones (see e.g., Mata,1997, Toxicol. Appl. Pharmacol. 144:189-197; Strauss-Soukup, 1997,Biochemistry 36:8692-8698; Samstag, 1996, Antisense Nucl. Acid Drug Dev.6:153-156).

“Amino acid” or “amino acid sequence” includes an oligopeptide, peptide,polypeptide, or protein sequence, or to a fragment, portion, or subunitof any of these, and to naturally occurring or synthetic molecules. Theterms “polypeptide” and “protein” include amino acids joined to eachother by peptide bonds or modified peptide bonds, i.e., peptideisosteres, and may contain modified amino acids other than the 20gene-encoded amino acids. The term “polypeptide” also includes peptidesand polypeptide fragments, motifs and the like. The term also includesglycosylated polypeptides. The peptides and polypeptides of theinvention also include all “mimetic” and “peptidomimetic” forms, asdescribed in further detail, below.

The term “isolated” includes a material removed from its originalenvironment, e.g., the natural environment if it is naturally occurring.For example, a naturally occurring polynucleotide or polypeptide presentin a living animal is not isolated, but the same polynucleotide orpolypeptide, separated from some or all of the coexisting materials inthe natural system, is isolated. Such polynucleotides could be part of avector and/or such polynucleotides or polypeptides could be part of acomposition, and still be isolated in that such vector or composition isnot part of its natural environment. As used herein, an isolatedmaterial or composition can also be a “purified” composition, i.e., itdoes not require absolute purity; rather, it is intended as a relativedefinition. Individual nucleic acids obtained from a library can beconventionally purified to electrophoretic homogeneity. In alternativeaspects, the invention provides nucleic acids, which have been purified,from genomic DNA or from other sequences in a library or otherenvironment by at least one, two, three, four, five, or more orders ofmagnitude.

As used herein, the term “recombinant” can include nucleic acidsadjacent to a “backbone” nucleic acid to which it is not adjacent in itsnatural environment. In one aspect, nucleic acids represent 5% or moreof the number of nucleic acid inserts in a population of nucleic acid“backbone molecules.” “Backbone molecules” according to the inventioninclude nucleic acids such as expression vectors, self-replicatingnucleic acids, viruses, integrating nucleic acids, and other vectors ornucleic acids used to maintain or manipulate a nucleic acid insert ofinterest. In one aspect, the (isolated, recombinant, enriched) nucleicacids represent 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%,61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, or more of thenumber of nucleic acid inserts in the population of recombinant backbonemolecules. “Recombinant” polypeptides or proteins refer to polypeptidesor proteins produced by recombinant DNA techniques; e.g., produced fromcells transformed by an exogenous DNA construct encoding the desiredpolypeptide or protein. “Synthetic” polypeptides or protein are thoseprepared by chemical synthesis, as described in further detail, below.

A promoter sequence can be “operably linked to” a coding sequence whenRNA polymerase which initiates transcription at the promoter willtranscribe the coding sequence into MRNA, as discussed further, below.

“Primer” includes either a single stranded polydeoxynucleotide or twocomplementary polydeoxynucleotide strands, which may be chemicallysynthesized. Such synthetic oligonucleotides have no 5′ phosphate andthus will not ligate to another oligonucleotide without adding aphosphate with an ATP in the presence of a kinase. A syntheticoligonucleotide can ligate to a fragment that has not beendephosphorylated.

All other terms are defined in the literature using Sambrook (Sambrooket al., 1989, “Molecular Cloning: A laboratory manual” Ed. 2, ColdSpring Harbor Press, Cold Spring Harbor) as a guide.

Genome Organization of the TSV

The TSV genome is a single-stranded RNA of positive polarity with a3′-poly(A) tail (Bonami et al., 1997, J. Gen. Virol. 78:313-319). Thegenome is 10205 nucleotides (nt) long with a 5′ untranslated region of377 nt and a 3′ untranslated region of 226 nt (Mari et al. 2002). Thereare two open reading frames (ORFs) in the TSV genome. ORF1 is 6324 ntlong, and encodes a 2107 amino acid (aa) polyprotein with a molecularmass of 234 kDa. ORF2 is 3036 nt long and encodes a 1011 aa polypeptidewith a molecular mass of 112 kDa (Mari et al., 2002, J. Gen. Virol.83:915-926; Robles-Sikisaka et al., 2001, Arch. Virol. 146:941-952).There is an intergenic region of 226 nt between the two ORFs. ORF1encodes non-structural proteins (helicase, a protease and aRNA-dependent RNA polymerase, RdRp), and ORF2 encodes the virionstructural proteins (FIG. 1). TSV virions contain three majorpolypeptides, designated as VP1, VP2, and VP3 (55, 40, and 24 kDa), andone minor polypeptide (58 kDa), designated as VPO (Bonami et al., 1997,J. Gen. Virol. 78:313-319). The N-termini of VP1 to VP3 have beensequenced, and the order of these proteins in ORF2 was found to be VP2,VP1 and VP3 (Mari et al., 2002, J. Gen. Virol. 83:915-926). TheN-terminal sequence of VPO has not been determined, but it has beenhypothesized that it might be processed from ORF2 in a manner similar toPSIV, an insect picorna-like virus infecting the brown-winged green bug,Plautia stali (Sasaki et al., 1998, Virology 244:50-58). The VP2/VP1cleavage site in TSV is conserved in insect picornaviruses: TSV(GF↓SKD), Plautia stali intestine virus, PSIV (GF↓SKP), Drosophila Cvirus, DCV (GF↓SKP) and Rhophalosiphum padi virus, RHPV (GW↓SKP)(Robles-Sikisaka et al., 2001, Arch. Virol. 146:941-952). The presumedVP1 and VP3 cleavage site in TSV (H↓A) is partially conserved with thoseused by insect picomaviruses Q↓(A,S,V) (Robles-Sikisaka et al., 2001,Arch. Virol. 146:941-952).

Comparison of TSV Genome Organization with Insect and MammalianPicornaviruses.

Picornaviruses have been isolated from a wide range of insect species.Based on their biological, biophysical properties, and genomeorganization data these viruses were classified as a member of a newlydesignated group, “Cricket paralysis-like viruses” in Picornaviridaewith CrPV as the type species of this group (Christian et al., 1998, In:Miller et al., Eds., “The Insect Viruses” New York, Plenum Press, pp.301-329; van Regenmortel et al., 2000, In: “Virus Taxonomy: TheClassification and Nomenclature of Viruses. The Seventh Report of theInternational Committee on Taxonomy of Viruses” Academic Press, SanDiego). Genomes of a number of these viruses have now been sequenced.These include CrPV (GenBank accession number AF218039), DCV (AF014388),acute bee paralysis virus (ABPV, NC002548); black queen cell virus(BQCV, AF183905) and SBV (AF092924) of honeybees; RhPV (AF022937), PSIV(AB006531), TrV (AF178440), HiPV (AB017037); IFV (AB000906) and TSV ofshrimp (AF277675). Among these viruses, the genome organizations of IFVand SBV were found to be similar to that of mammalian picornaviruses.They contain a single long ORF with the capsid proteins located at theN-terminal end and the non-structural proteins at the C terminal end. Incontrast, the genomes of CrPV, DCV, RhPV, PSIV, HiPV, TrV, and TSVcontain two long ORFs (ORF1 and ORF2) separated by an intergenic region.For all but RhPV, the length of the intergenic region varies from171-207 nucleotides. The intergenic region of RhPV is 525 nucleotideslong. The 5′ end of ORF1 contains the non-structural proteins and the 3′end of ORF2 contains the capsid proteins (FIG. 1). All of these virusesshow greater sequence similarity to each other than with any of themammalian picornaviruses.

EXAMPLES

The invention, as contemplated herein, is a composition comprisingnucleotide sequence of an insect picornavirus (IPV) such as the Taurasyndrome virus (TSV), infectious hypodermal and hematopoietic necrosisvirus (IHHNV), or white spot syndrome virus (WSSV), and one or morecistronic elements (e.g., a shrimp gene sequence) operably linked therewith. Alternatively, the invention provides methods of protection ofanimals, in particular crustaceans from disease and manipulation ofanimals, specifically crustaceans, in vitro and in vivo. The inventionprovides tools critical for the production of an immortalized shrimpcell line. The following examples describe some aspects of the inventionand are used for exemplification purposes only and are not intended tolimit the scope of the invention in any way.

Example 1

Identification of Internal Ribosomal Entry Site (IRES) Elements in TSVand Insect Picorna-Like Viruses.

Insect picornaviruses with dicistronic genomes have two unique features.First, no sub-genomic RNA is produced for translation of the capsidproteins. Second, the coat protein cistron appears to lack an initiatingmethionine suggesting that the coat protein is translated throughinternal initiation mediated by an internal ribosomal entry site (IRES).

Functional IRES elements have been identified in the intergenic regionof CrPV and PSIV (Wilson et al., 2000, Mol. Cell. Biol. 20:4990-4999;Sasaki et al., 1999, J. Virol. 73:1219-1226), and homologous sequenceshave been identified in TSV (Mari et al., 2002, J. Gen. Virol.83:915-926, FIG. 2). Cap-independent translation in PSIV ORF2 has beendemonstrated in vitro using a rabbit reticulocyte lysate (Sasaki et al.,2000, Proc. Natl. Acad. Sci. USA 97:1512-1515). In CrPV, the initiationcodon for IRES mediated translation was identified as CCU, whereas, inPSIV and RhPV the initiation codon was found to be CUU. It has beenshown that the CCU/CUU triplets are part of the inverted repeat sequenceof the IRES elements which form RNA pseudoknot structures essential forIRES activity (Wilson et al., 2000, Mol. Cell. Biol. 20:4990-4999;Sasaki et al., 1999, J. Virol. 73:1219-1226). In TSV, although there isan in-frame methionine in ORF2, N-terminal sequencing of the VP2 capsidprotein identified an alanine at the terminal position in the sequencedprotein (ANPVEIDNFDTT, SEQ ID NO: 17; Mari et al. 2002, Refugio et al.,2001). The alanine codon is preceded by both a proline (CCU) and amethionine (AUG) codon (MPANPVE, SEQ ID NO: 18). For methionine to bethe initiation codon for TSV ORF2, MP residues would need to be removedfrom the mature protein. Such post-translational processing has neverbeen found in eukaryotes and it is likely that TSV employs anIRES-mediated cap-independent mechanism for translation of thestructural proteins similarly to the insect picomaviruses.

Multiple alignments of the intergenic regions of insect picorna-likeviruses are shown in FIG. 2. The nucleotide difference in the intergenicregion among insect picoma-like viruses was calculated using thecomputer program MEGA (Kumar et al., 2001, Molecular EvolutionaryGenetic Analysis (MEGA) software, version 2.0, Institute of MolecularEvolutionary Genetics, Arizona State University, Ariz., USA) (Table 1).The program calculates the p-distance (nucleotide), which is defined asthe proportion (p) of nucleotide sites at which the two sequencescompared are different. It is obtained by dividing the number ofnucleotide differences by the total number of nucleotides compared.

The nucleotide residues that constitute the stem loop structure rightbefore the ORF2 is indicated in FIG. 2C. This region in TSV can formsecondary structures like other insect picorna-like viruses, andinitiate IRES-mediated cap-independent translation of TSV ORF2 thatencodes capsid proteins.

In insect picorna-like viruses like DCV, it has been shown thatstructural proteins are produced in vast excess over non-structuralproteins in virus-infected cells (Moore et al., 1980, J. Virol. 33:1-9;Moore et al., 1981, Arch. Virol. 68:1-8). This is in contrast to humanpicomavirus-infected cells, wherein approximately equimolar amounts ofstructural and non-structural proteins are produced (Ruckert, 1996, InFields et al., Eds., “Field Virology” Lippincott-Raven, Philadelphia,Pa., pp. 609-654). The IRES-mediated translation of the coat proteins ininsect picorna-like viruses with dicistronic genome, therefore, providesa mechanistic explanation for the abundance of structural compared tonon-structural proteins in insect picorna-like virus infected cells.Insect picorna-like viruses with dicistronic genomes encode two distinctpolyproteins: non-structural proteins encoded by the upstream ORF andthe structural protein encoded by the downstream ORF. Translation of thetwo ORFs seems to be independently controlled. This is in contrast tothe picomaviruses with single ORF genomes, which are translated into asingle polyprotein and post-translationally processed to give rise toboth structural and non-structural proteins (Ruckert, 1996, In Fields etal., Eds., “Field Virology” Lippincoft-Raven, Philadelphia, Pa., pp.609-654).

All picomaviruses that infect mammalian, insect, or plant hosts, containa 5′-untranslated region. Like the IRES element in the intergenic regionof insect picoma-like viruses with dicistronic genome, the 5′untranslated region of mammalian picornaviruses were shown to containsecondary structures that serve as an IRES element and thus, capable ofcap-independent translation.

TABLE 1 Pair-wise comparisons of the intergenic regions of insectpicorna-like viruses using the program MEGA. CrPV DCV PSIV HiPV TrV RhPVTSV BQCV ABPV CrPV DCV 0.200 PSIV 0.408 0.438 HiPV 0.385 0.446 0.331 TrV0.369 0.385 0.415 0.431 RhPV 0.369 0.369 0.462 0.485 0.346 TSV 0.5460.523 0.600 0.615 0.562 0.515 BQCV 0.292 0.323 0.400 0.446 0.392 0.4150.562 ABPV 0.446 0.431 0.569 0.592 0.531 0.538 0.585 0.485

Example 2

Genome Organization of IHHNV

The IHHNV genome contains a single stranded linear DNA of 4.1 kb (Bonamiet al., 1990). Almost the entire IHHNV genome except the 5′-and3′-terminal ends has been sequenced (Shike et al., 2000, Virology,277:167-177). There are three large open reading frames (ORFs) in theIHHNV genome (FIG. 3). The middle and the right ORFs are present in theplus 3 frame, whereas the left ORF is present in the plus 3 frame. Themiddle ORF completely overlaps with the left ORF, whereas the right ORFpartially overlaps with the left ORF. The left, middle and the rightORFs have potential coding capacities of 666 amino acids (75.77 kDa),363 aa (42.11 kDa) and 329 aa (37.48 kDa), respectively.

The left ORF most likely encodes for a major non-structural protein(NS-1), which contains replication initiation motifs, NTP-binding, andhelicase domains. The right ORF encodes the capsid protein. The aasequence of middle ORF does not show any similarity with databaseentries, and therefore, the putative function of this ORF is unknown.There are two potential promoters identified in IHHNV genome. One calledP2 (based on map unit 2) is located upstream of the left ORF, and theother, called P61, is located upstream of right ORF (nucleotide position2398-2448). Overall, the genome organization revealed that IHHNV isclosely related to densoviruses of the genus Brevidensovirus in thefamily Parvoviridae (Shike et al., 2000, Virology, 277:167-177). Aschematic representation of the genome organization of IHHNV genome isshown in FIG. 3.

Example 3

Amplification of IHHNV P61 Promoter by Polymerase Chain Reaction (PCR)

In order to evaluate the promoter activity of IHHNV P61 promoter, a 166bp DNA flanking the P61 promoter was designed. The sequence of theprimers were: IHHNP61F: 5′-GGTAC CTCCA GCTGA TGGTA AAGCT-3′ (SEQ ID NO:19; nucleotide position 2346-2371 in AF273215) and IHHNP61R: 5′-TTCGTATTCT TGGAA GAGTC CTAG3″ (SEQ ID NO: 20; nucleotide position 2488 inAF273215). The reaction mixture for the PCR included 1× Promegathermophilic DNA polymerase buffer, 2.0 millimolar MgCl₂, 0.4 micromolardNTPs, 0.8 micromolar of forward and reverse primer, 7.5 units ofPromega Taq DNA polymerase, and 100 nanograms of total genomic DNAisolated from IHHNV-infected shrimp in a 25 microliter reaction volume.The thermal profile for the PCR was 95 degrees Celsius for 5 minutesfollowed by 40 cycles of 95 degrees Celsius 30 seconds, 52 degreesCelsius 30 seconds, and 72 degrees Celsius 1 minute before cooling at 4degrees Celsius. PCR amplified DNA was run in a 1.5% agarose gelcontaining ethidium bromide using Tris-acetate EDTA buffer andphotographed. A photograph of the agarose gel is shown in FIG. 4. Theamplified DNA was gel purified using Qiagen gel purification kit(Qiagen, California), and sequenced using IHHNVP61F primer. Thenucleotide sequence of the amplified DNA showed 100% similarity with theGenBank database entry, AF273215.

Example 4

WSSV Genome Organization

The genome of WSSV contains a circular double-stranded (ds) DNA of about300 kb in size. So far three geographic isolates of WSSV have beencompletely sequenced including a Thai-isolate (292,967 bases, GenBankaccession number AF369029, van Hulten et al., 2001, Virology 286:7-22),a Chinese isolate (305,307 bases, Yang et al., 2001, J. Virol.75:11811-11820; GenBank accession number AF332093), and an isolate fromTaiwan (307,287 bases, GenBank accession number AF440570). Arepresentative genetic map of one of the isolates, Thai-isolate, isshown in FIG. 5. The three isolates show an overall nucleotide identityof 99.32% (Marks et al., 2004, Arch. Virol. 149:673-697). There are 181to 184 predicted open reading frames (ORFs) in the WSSV genome dependingon the isolates and only 6% of the ORFs have putative homologues amongthe GenBank database entries (van Hulten et al., 2001, Virology286:7-22). The size differences among the isolates are due to a 13210 bpdeletion in the Thai-isolate and a 1168 bp deletion in the WSSV Chineseisolate compared to the WSSV Taiwan isolate (Marks et al., 2004, Arch.Virol. 149:673-697).

WSV is morphologically similar to insect baculovirus. However,phylogenetic analysis of ribonucleotide reductase and protein kinasegenes revealed that WSV does not share a common ancestor withbaculoviruses (van Hulten et al., 2000, J. Gen. Virol. 81:307-316; vanHulten et al., 2001b, Virus Genes 22:201-207). Due to its limitedsequence similarity with other viruses sequenced so far, WSSV has beenplaced in a new virus family, Nimaviridae, genus Whispovirus, asdescribed in the art, for example in the NCBI's publicly-available website relating to viral taxonomy.

Example 5

Transposable Elements in WSSV Genome

One of the three isolates of WSSV, the Taiwan isolate, contains a genethat encodes a transposase. The WSSV Taiwan isolate transposase geneshows 100% similarity with the homologous transposable elements ofprokaryotic and eukaryotic origin. The regions upstream and thedownstream of the ORF contains inverted repeats, a common feature oftransposable elements. At the site of insertion of the transposableelement in the WSSV genome, there has been a duplication of the WSSVsequence. The similarity of WSSV transposase with the homologoussequence in the GenBank database, and the genetic map of the transposasegene in WSSV genome are shown in FIGS. 6 and 7.

Example 6

Identification of a Drosophila Homologue of Penelope TransposableElement in Shrimp

Transposable elements (TEs) have been identified from a wide variety ofspecies including bacteria, plants, invertebrates, and vertebrates, andthey constitute a considerable portion of the genetic makeup of aspecies. For example, TEs make up 3% of yeast genome (Kim et al., 1998),10-15% of Drosophila sp. genome (SanMiguel et al., 1996, Science274:765-768), and about 50% of maize genome (Pimpinelli et al., 1995).In human, TEs constitute 43% of the genome representing 4.3 million TEs(Smit, 1999, Curr. Opin. Genet. Dev. 9:657-663; Li et al., 2001, Nature409:847-849). Although TEs have long been held as “selfish DNA”, onlyrecently the importance of TEs is being realized (Kumar and Hirochika,2001; Plasterk et al, 1999). TEs have been used in linkage mapping,genetic fingerprinting, molecular breeding, transgenic research, andevolutionary studies (Kumar et al., 2001, Trend Plant Biol. 6:127-134;Plasterk et al., 1999, Trends Genet.15:326-332).

Dhar and colleagues have recently reported the construction of a sizefractionated genomic DNA library from shrimp (Penaeus monodon) forisolating microsatellite sequences for genetic analysis (Xu et al.,1999, Animal Genet. 30:1-7; Xu et al., 2004, J. Biochem. Genet., Inpress). GenBank database search for one of the DNA clones (GenBankaccession number AF077579) showed significant similarity to a Drosophilavirilis non-LTR transposable element Penelope (FIG. 8). The activationof Penelope elements in Drosophila has shown to be associated with asyndrome of aberrant traits collectively known as dysgenesis (Kidwell etal., 1997, Proc. Natl. Acad. Sci. USA 94:7704-7711). Intact Penelopeelements of Drosophila encode a reverse transcriptase and anendonuclease of the UvrC type, which may play a role in Penelopeintegration (Lankenau et al., 1997, Proc. Natl. Acad. Sci. USA94:196-201). Recently, Pyatkov et al. (2002, Proc. Natl. Acad. Sci. USA99:16150-16155) used Penelope element as a transformation vector forgerm-line transformation of Drosophila. Penelope was found to beactively transcribed, and undergoes massive copy number increase only intransformed lines where transformation was performed using a full-lengthcopy as opposed to a truncated copy of the Penelope element.

Example 7

Construction of a Shrimp Transient Expression Vector

In order to construct a shrimp transient expression vector, IHHNV P61promoter was amplified from IHHNV-infected shrimp using genomic DNA astemplate for the PCR (FIG. 4). The PCR amplified DNA showed 100%identity to IHHNV genomic sequence (GenBank accession number AF273215).The IHHNV P61 promoter is cloned into a TOPO vector followingmanufacturer's protocol (Invitrogen, Inc., California).

The entire TSV intergenic region between ORF1 and ORF2 is amplified byreverse transcriptase-polymerase chain reaction (RT-PCR) followingpublished protocol (Dhar et al., 2002). The cDNA amplicon is cloned intoTOPO vector (Invitrogen, Inc, California) and recombinant clones aresequenced to confirm the identity of the cDNA. The primer sequences foramplifying the TSV intergenic region are: Forward 5′-TAGCA CCACC CGATCGTAAA C-3′ (SEQ ID NO: 21), Reverse 5′-TAATT AAGTC CCACC ACGCA AG-3′(SEQ ID NO: 22).

After confirming the sequence, the insert for the TSV intergenic regionis digested from the recombinant clone with appropriate restrictionenzyme, run in a 1.5% agarose gel following standard protocol (Sambrooket al., 1989, “Molecular Cloning: A laboratory manual” Ed. 2, ColdSpring Harbor Press, Cold Spring Harbor) and gel purified using theQiagen gel purification kit (Qiagen, California).

The TSV intergenic region is cloned downstream of the IHHNV promoter.For this, the plasmid DNA from a recombinant clone containing the IHHNVpromoter is digested with enzymes compatible with the TSV intergenicregion insert and ligated using T4 DNA ligase and cloned in E. colifollowing standard protocol (Sambrook et al., 1989, “Molecular Cloning:A laboratory manual” Ed. 2, Cold Spring Harbor Press, Cold SpringHarbor).

The recombinant clones containing the IHHNV P61 promoter and the TSVintergenic region are identified by colony PCR using IHHNVP61F primerand a vector derived primer. Clones that contain appropriate orientation(see FIG. 9A) are taken for subsequent sub-cloning into a pDsRed vector(BD Bioscience, California). The plasmid DNA containing the IHHNV P61promoter and the TSV intergenic region are digested with appropriateenzyme, and cloned upstream of selectable marker DsRed in pDsRed vector(BDClontech). The plasmid DNA of the recombinant clones is sequenced toconfirm the identity of the transformants. A schematic representation ofshrimp transient vector is presented in FIG. 9A.

Example 8

Assay of Shrimp Transient Expression In Vivo

Plasmid DNA from recombinant shrimp expression vector is injected intothe tail muscle of juvenile shrimp (about 1 gram) and the activity ofthe reporter gene (DsRed) is assayed either directly using atransilluminator and direct observation of the shrimp or by excision ofthe area injected and then extraction of soluble protein and analysis ona fluorescent microplate reader (Matz et al., 1999, Nat. Biotechnol.17:969-973). Shrimp injected with the plasmid DNA of a non-recombinantclone serve as a control. Injected shrimp are sacrificed at 24, 48, 72hr post injection and the levels of DsRed protein is measured. Afluorescent microplate reader is used for this analysis.

Tissue is homogenized with a tissue homogenizer in 50 millimolar Tris(pH 7.0) buffer plus proteinase inhibitor (1 millimolarphenylmethylsulfonyl fluoride, 5 millimolar benzamidine). The solubleportion is recovered after a brief centrifugation. This is analyzed fortotal protein by the Lowry method and aliquotted into black Nuncmicroplates in serial dilution. Fluorescence is read on SpectraFluorPlus (Tecan) reader at 485 nm excitation and 535 emissions. Controls arerun with standard DsRed protein either made internally or obtainedcommercially.

Example 9

Assay of Shrimp Transient Expression In Vitro

Plasmid DNA from recombinant shrimp expression vector is injected totransfect primary cell culture of shrimp using lipofecting agent. Cellstransfected with the plasmid DNA of non-recombinant clone serve as acontrol. The activity of the reporter gene (DsRed) is assayed at 24, 48,72 hours post transfection following standard protocol (see Example 8).

Example 10

Cloning of a Full-Length Shrimp Penelope Element

In order to clone a full-length shrimp Penelope element, RT-PCR isperformed using DNase treated total RNA from Penaeus vannamei shrimp toamplify a 500 bp amplicon using shrimp Penelope primers. The primers aredesigned based on the partial sequence of shrimp Penelope elementavailable in the GenBank database (accession number AF077579). TheRT-PCR protocol for amplifying the Penelope element is same as describedfor amplifying other shrimp genes like beta-actin (Dhar et al., 2002).The amplified cDNA is run in a 1.5% agarose gel following standardprotocol (Sambrook et al., 1989, “Molecular Cloning: A laboratorymanual” Ed. 2, Cold Spring Harbor Press, Cold Spring Harbor) and gelpurified using Qiagen gel purification kit (Qiagen, California). Thegel-purified cDNA is cloned in to a TOPO vector following manufacturer'srecommendations (Invitrogen, Inc., California). Recombinant clones aresequenced to confirm the identity. The 500 bp partial clone of shrimpPenelope element is used as a probe to pull out a full-length clone ofPenelope element from a shrimp (Penaeus vannamei) cDNA library (Dhar etal., 2004, “Comparison of expression profiles of healthy and white spotsyndrome virus (WSSV) infected shrimp (Penaeus vannamei) by expressedsequence tag (EST) analysis,” Presented at the World Aquaculture Societymeeting, Shrimp Genomics Section, March 1-5, 2004, Honolulu, Hi.). Ifthe isolated clone lacks 5′-end, 5′-RACE technique is employed to clonethe 5′-terminal sequences following protocol used to clone the5′-terminal sequence of other shrimp gene (Roux et al., 2002, J. Virol.76:7140-7149). Recombinant clones are sequenced to confirm the identityof the clone.

Example 11

Construction of a Shrimp Transfection Vector Using a Shrimp PenelopeElement

In order to construct a transfection vector, IHHNV P61 promoter (FIG. 4)is cloned into TOPO vector following manufacturer's recommendations(Invitrogen, Inc., California). The entire shrimp Penelope element ORFis amplified from the recombinant clone by PCR. The primers for the PCRare based on the sequence of the Penelope element ORF. The primersequences contain unique restriction enzyme sites that are need to cloneit downstream of IHHNVP61 promoter. Amplified cDNA is digested withthose enzymes before cloning downstream of IHHNVP61 promoter in TOPOvector. Ligation and transformation are done following standard protocol(Sambrook et al., 1989, “Molecular Cloning: A laboratory manual” Ed. 2,Cold Spring Harbor Press, Cold Spring Harbor).

The entire TSV intergenic region (TSV IRES) between ORF1 and ORF2 isamplified by reverse transcriptase-polymerase chain reaction (RT-PCR)following published protocol (Dhar et al., 2002, J. Virol. Meth.104:69-82). The cDNA amplicon is cloned into TOPO vector (Invitrogen,Inc, California) and recombinant clones are sequenced to confirm theidentity of the cDNA. The primer sequences for amplifying the TSVintergenic region are: Forward: 5′-TAGCACCACC CGATCGTAAA C-3′ (SEQ IDNO: 23), Reverse 5′-TAATTAAGTC CCACCACGCA AG-3′ (SEQ ID NO: 24). Afterconfirming the sequence, the insert for TSV IRES is digested from therecombinant clone with appropriate restriction enzyme, run in a 1.5%agarose gel following standard protocol (Sambrook et al., 1989,“Molecular Cloning: A laboratory manual” Ed. 2, Cold Spring HarborPress, Cold Spring Harbor) and gel purified using Qiagen gelpurification kit (Qiagen, California). The TSV intergenic region issub-cloned upstream of DsRed open reading frame in pDsRed vector (BDBioscience, California). The recombinant clones containing the TSVintergenic region is sequenced to confirm the orientation and identityof the clones.

The TSV IRES-pDsRed plasmid is digested with two enzymes that cutupstream of the TSV IRES element. Similar enzymes are used to cut outthe IHHNVP61-Penelope construct. The IHHNVP61-Penelope insert is gelpurified using Qiagen gel purification kit before cloning upstream ofthe TSV intergenic region in pDsRed-TSV Intergenic construct. All enzymedigestion, ligation and transformation are done following standardprotocol (Sambrook et al., 1989, “Molecular Cloning: A laboratorymanual” Ed. 2, Cold Spring Harbor Press, Cold Spring Harbor).Recombinant clones are sequenced to confirm the orientation and identityof the inserts.

Finally, the inverted terminal repeat elements of shrimp Penelope areamplified from shrimp full-length Penelope gene and cloned upstream ofIHHNVP61 promoter and downstream of the selectable maker gene (in thiscase DsRed). A schematic representation of the final shrimp transfectionvector is presented in FIG. 9B.

Example 12

Construction of a Shrimp Transfection Vector Using a WSSV TransposableElement

In order to construct a transfection vector, IHHNV P61 promoter (FIG. 4)is cloned into a TOPO vector following manufacturer's recommendations(Invitrogen, Inc., California). The entire shrimp WSSV transposase ORFis amplified by RT-PCR using DNase-treated total RNA from WSSV-infectedshrimp tail muscle. The primers for the PCR are based on the sequence ofthe WSSV transposase ORF (GenBank accession number AF440570). The primersequences contain unique restriction enzyme sites that are needed toclone it downstream of P61 promoter. Amplified cDNA is digested withthose enzymes before cloning downstream of IHHNVP61 promoter in TOPOvector. Ligation and transformation are done following standard protocol(Sambrook et al., 1989, “Molecular Cloning: A laboratory manual” Ed. 2,Cold Spring Harbor Press, Cold Spring Harbor). Recombinant clones aresequenced to confirm the identity of the WSSV transposases clones.

The entire TSV intergenic region (including the TSV IRES) between ORF1and ORF2 is amplified by reverse transcriptase-polymerase chain reaction(RT-PCR) following published protocol (Dhar et al., 2002, J. Virol.Meth. 104:69-82). The cDNA amplicon is cloned into a TOPO vector(Invitrogen, Inc, California) and recombinant clones are sequenced toconfirm the identity of the cDNA. The primer sequences for amplifyingthe TSV intergenic region are: Forward: 5′-TAGCACCACC CGATCGTAAA C-3′(SEQ ID NO: 25), Reverse 5′-TAATTAAGTC CCACCACGCA AG-3′ (SEQ ID NO: 26).After confirming the sequence, the insert for TSV IRES is digested fromthe recombinant clone with appropriate restriction enzyme, run in a 1.5%agarose gel following standard protocol (Sambrook et al., 1989,“Molecular Cloning: A laboratory manual” Ed. 2, Cold Spring HarborPress, Cold Spring Harbor) and gel purified using Qiagen gelpurification kit (Qiagen, California). The TSV intergenic region issub-cloned upstream of DsRed open reading frame in pDsRed vector (BDBioscience, California). The recombinant clones containing the TSVintergenic region are sequenced to confirm the orientation and identityof the clones.

The TSV IRES-pDsRed plasmid is digested with two enzymes that cutupstream of the TSV IRES element. Similar enzymes are used to cut outthe IHHNVP61-WSSV transposase construct. The IHNVP61-WSSV transposase isgel purified using Qiagen gel purification kit before cloning upstreamof the TSV intergenic region in pDsRed-TSV Intergenic construct. Allenzyme digestion, ligation and transformation are done followingstandard protocol (Sambrook et al., 1989, “Molecular Cloning: Alaboratory manual” Ed. 2, Cold Spring Harbor Press, Cold Spring Harbor).Recombinant clones are sequenced to confirm the orientation and identityof the inserts.

Finally, the inverted terminal repeat elements of WSSV are amplified byRT-PCR and cloned upstream of IHHNVP61 promoter and downstream of theselectable maker gene (in this case DsRed). A schematic representationof the final shrimp transfection vector is presented in FIG. 10.

Example 13

Transformation of shrimp primary cell culture with a transfection vectorand assay of expression of recombinant protein in vitro

Plasmid DNA from a recombinant shrimp transfection vector is used totransfect primary cell culture of shrimp using a lipofecting agent.Shrimp primary cell culture for hemocytes is made following a publishedprotocol (e.g., Itami et al., 1999, Meth. Cell Sci. 21:237-244). Shrimphemocyte primary culture is transfected with the plasmid DNA of shrimptransfection vector described in Example 11, FIG. 9B. The IHHNVP61 or P2promoters driving Shrimp Penelope full-length ORF serve as a control.Alternative control promoters are available and are employed as needed(e.g., human cytomegaly virus (Tseng et al., 2000, Theriogenology54:1421-1432) and pantropic retroviral promoters (Bums et al., 2000, PCTPublication WO00/75288; Shike et al., 2000, Virology, 277:167-177)). Theactivity of the reporter gene (DsRed) is assayed at 24, 48, 72 hourspost transfection following the procedure outlined in Example 8.

In order to confirm the shrimp Penelope mediated integration of markergene (DsRed), total genomic DNA is isolated from the shrimp transfectedcells (primary cell lines) using Qiagen DNA extraction kit (Qiagen,California), digested with restriction enzymes, run in an agarose geland blotted on to Nylon membrane (Sambrook et al., 1989, “MolecularCloning: A laboratory manual” Ed. 2, Cold Spring Harbor Press, ColdSpring Harbor). Southern hybridization is performed using TSV IRES probeand DsRed probe (Sambrook et al., 1989, “Molecular Cloning: A laboratorymanual” Ed. 2, Cold Spring Harbor Press, Cold Spring Harbor). Positivehybridization signals using these probes indicate transformation of theshrimp DNA with the transfection vector construct.

Example 14

Diagnostic Use of the Transgenic Cell Line of Example 13.

Adherent shrimp cells produced as in Example 13 are utilized for thediagnosis of viral disease by growth of the cells in a microplate. Thesecells are then exposed to shrimp tissue to be analyzed that has beenmacerated in a sterile tube and serially diluted using standardtechniques. As infection proceeds, the fluorescence of the DsRed ismonitored in a SpectraFluor Plus plate reader as described in Example 8.Cell mortality due to viral infection is reflected in a decrease inDsRed emission and viral titer can be determined rapidly versus astandard curve run simultaneously.

Example 15

Construction of a IHHNV p61 and p2 Promoter Driven Luciferase Vectors.

The IHHNV p61 promoter element (GenBank accession # AF273215) wasexcised from a previously constructed and verified clone displayed inFIG. 11 using PlasMapper software (Dong et al., 2004, Nucl. Acids Res.32(Web Server issue):W660-664).

Briefly, the targeted fragment was PCR amplified using the forwardprimer p61 1 F (5′-TAC AGA GCT CGG TAC CTC CAG CTG A-3′; SEQ ID NO: 27)and the reverse primer p61 1 R (5′-GCT AGC TAG CTT CGT ATT CTT GGA AGAGTC-3′; SEQ ID NO: 28). This amplicon was then digested with SacI andNheI restriction enzymes (underlined in primer sequences) for insertion.The digested amplicon was gel purified using methods described forMiniElute Gel Extraction Kit in the manufacturer's instructions (Qiagen,Valencia, Calif.). The purified fragment was then inserted into apGL3-Basic reporter vector containing a modified coding region forfirefly (Photinus pyralis) luciferase (Promega Corp. Madison, Wis.) thathad been digested and gel purified as above (FIG. 12).

Replicate clones were verified for sequence integrity at an off-sitelocation using standard methods applied to an ABI 3100 capillary DNAanalyzer (Applied Biosystems, Foster City, Calif.). The verifiedsequence was then excised out as before and inserted into the samelocation within a vector containing sequences reported to enhanceexpression of cloned fragments (pGL3-Enhancer, GenBank accession #U47297 (Cat# E1771, Promega Corp. Madison, Wis.). Again, replicateclones were verified as stated above. This produced the construct shownin FIG. 13.

The p2 promoter element from IHHNV was synthesized using methodsdescribed previously (Smith et al., 2003, Proc. Natl. Acad. Sci. USA100:15440-15445) at a commercial facility. Briefly, the synthesizedfragment was inserted into the general use cloning vector pUC19 (GenBankaccession # L09137). That plasmid was then sequence verified at the samelocation. Using the synthesized SacI and NheI sequences for excision,the p2 fragment was removed and inserted into the pGL3-Enhancer vectormentioned above. Replicate clones were verified as above. This producedthe construct shown in FIG. 14.

Example 16

Construction of IHHNV p2 and p61 Promoter Driven DsRed2 Reporter PlasmidConstruction

The IRES element from TSV (Cevallos et al., 2005, J. Virol. 79:677-683;GenBank accession # AF277675) was excised from a previously constructedand verified clone. Briefly, the targeted fragment was PCR amplifiedusing the forward primer TSV IGR SACF (5′-GAT CGA GCT CTA GCA CCA CCCGAT CGT AAA C-3′; SEQ ID NO: 29) and TSV IGR BAMR (5′-GAT CGG ATC CTAATT AAG TCC CAC CAC-3′; SEQ ID NO: 30). This amplicon was then digestedwith SacI and BamHI restriction enzymes (underlined in primer sequences)for insertion. The digested amplicon was gel purified using standardmethods. The purified fragment was then inserted into a previouslyconstructed and verified reporter vector pSTD (FIG. 15A) containing amodified fluorescent protein to provide the construct shown in FIG. 15B.Replicate clones of the above were verified by sequencing.

An extended version of the TSV IGR was produced for cloning into theluciferase reporter vectors from above. The targeted fragment was PCRamplified using the forward primer TSV-IGR 2 F (5′-ATA CTC GAG AAC TAATAG CAC CAC CCG-3′; SEQ ID NO: 31) and TSV-IGR 2 R (5′-TCC AAG CTT TTGTTG TAT CAA AAT TAT -3′; SEQ ID NO: 32), and electrophoresed in a 1.5%agarose gel (FIG. 16).

Example 17

Transient Expression of a Marker Protein in Shrimp.

Shrimp Handling.

Naive juvenile shrimp (Penaeus vannamei) free of specific pathogens wereobtained from an outside source (Penaeus(Litopenaeus) vannamei (Konaline), about 1 gram, obtained from The Oceanic Institute. Waimanalo,Hi.). Specific Pathogen-Free (SPF) shrimp were free of those pathogenscited in Lightner (2003, “Exclusion of Specific Pathogens for DiseasePrevention in a Penaeid Shrimp Biosecurity Program,” In “Biosecurity inAquaculture Production Systems: Exclusion of Pathogens and OtherUndesirables” The World Aquaculture Society, Baton Rouge, La.). Theanimals were held in artificial seawater at about 26 degrees Celsiuscontaining standard sea salt components (INSTANT OCEANS® Cat# SS1-160P,Aquarium Systems Inc., Mentor, Ohio) until use. Shrimp Grower feed (2.4millimeter 3/32, Cat# SI-35, Zeigler Bros., Inc. Gardners, Pa.) wasgiven at regular intervals with the amount being about 3% of the totalbody weight. Excess feces and uneaten food were aspirated from the tankswhen needed.

Shrimp were then distributed into 5 liter tanks (STERILITE® SHOW OFFS™,UPC#073149894366, Sterilite Corp. Townsend, Mass.) containing 4 litersof artificial seawater as above. Each group (two groups of 6 and fourgroups of 7) was handled according to the procedures above untilexperiment termination.

DNA Injection.

Using a 1 milliliter syringe, 50 microliters of solution (20% Glycerol,0.9% NaCl, about 10 micrograms of purified plasmid DNA) was injectedjust under the carapace into the tail muscle of the animals. Theinjection site of each animal was the same. Animals were sacrificedafter about 72 hours and screened for the presence of luciferase.

Tissue Lysate.

Animals were removed from the tanks and chilled in ice-cold water forabout 5 minutes to anesthetize them. The tissue surrounding theinjection site was excised and placed into a tube of standard lysisreagent supplemented with components for stabilization (1× Cell CultureLysis Reagent; Cat# E153A, Promega Corp., Madison, Wis. 1× ProteaseInhibitor Cocktail; Cat# P-2714, Sigma, St. Louis, Mo.). The excisedtissue was homogenized with a pestle (1.5 milliliter microcentrifugetube pestles; Cat# 01-1415-5390, USA/Scientific Plastics®, MiltonKeynes, England) to release total cellular protein content intosolution. The samples were then clarified through centrifugation and thesupernatant removed and placed in a clean tube. The samples were held at4 degrees Celsius until use.

Luciferase Assay.

All reactions listed here were performed in microplate format (96-well,white bottom; Cat# 655083, Greiner bio-one, Inc., Longwood, Fla.) usingstandardized reagents (BRIGHT-GLO™ luciferase assay substrate; Cat#E263A, Promega Corp., Madison, Wis.) and the luminescent signalquantified using a luminometer (SPECTRAFluor PLUS; Firmware-V 6.0006-07-2003 Spectra; XFLUOR4 Version-V 4.50, Tecan US. Research TrianglePark, N.C.). The default settings for the luminometer were used. Allreactions were carried out in a total volume of 100 microliters (50microliters of sample +50 microliters of standardized reagent). Readingswere made as soon as possible after the addition of reagent.

A standard curve using recombinant luciferase (QUANTILUM® recombinantluciferase. Source: North American firefly, Photinus pyralis; Cat#E170B, Promega Corp. Madison, Wis.) was constructed beginning at aconcentration of 1 femtogram per microliter and serially diluted 2-foldto 15.6 attograms per microliter. Duplicate wells were averaged togetherfor producing the graph in FIG. 17.

Tissue lysate samples were read in triplicate wells. These technicalreplicate values were averaged together to form the chart in the datasection below. Biological replicate values lying 1 standard deviation(SD) outside of the mean of the entire group were excluded. Standardswere run to provide a plot of the standard curve values, shown in FIG.17.

Macerated tissue samples were treated as described above and assayed forluciferase expression. Shrimp luciferase data were extrapolated into theluciferase standard curve (Table 2). Luciferase activity was expressedas relative light units (RLU) for each biological replicate in theexperiment and normalized to the amount of tissue excised.

TABLE 2 Data from the luciferase assay comparing samples of shrimptissue excised from tail muscle injected with controls (Buffer Only orVector Only) or constructs containing IHHNV p2 promoter (pSTF1.1) or p61promoter (pSTH). Underlined values indicate outliers (Defined as thosesamples that lie 1 standard deviation outside of the group mean).Treatments RLU/100 mg Tissue Buffer Only 19.14 Buffer Only 31.67 BufferOnly 29.93 Buffer Only  6.62 Buffer Only 30.80 Buffer Only 13.95 pSTF1.141.50 pSTF1.1 42.26 pSTF1.1 41.96 pSTF1.1 33.09 pSTF1.1 26.69 pSTF1.157.86 pSTF1.1 78.57 pGL3-Enhancer 45.98 pGL3-Enhancer 24.16pGL3-Enhancer 13.42 pGL3-Enhancer 14.50 pGL3-Enhancer 32.56pGL3-Enhancer 41.18 pSTI1.1 411.49  pSTI1.1 357.96  pSTI1.1 25.00pSTI1.1 206.43  pSTI1.1 166.81  pSTI1.1 505.56  pSTI1.1 222.54  pSTH1.137.73 pSTH1.1 20.81 pSTH1.1 61.45 pSTH1.1 14.42 pSTH1.1 57.66 pSTH1.1119.09  pSTH1.1 41.75

TABLE 3 Eliminating the outliers from Table 2 and calculating the mean,SD and % CV (% CV—Coefficient of Variation, defined as (SD/mean) * 100).Treatment Mean SD % CV Buffer Only 27.88 5.87 21.06 pSTF1.1 43.34 8.9820.72 pGL3-Enhancer 32.63 8.51 26.08 pSTI1.1 222.54 105.66 47.48 pSTH1.143.88 16.38 37.33

The disclosure of every patent, patent application, and publicationcited herein is hereby incorporated herein by reference in its entirety.

While this invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention can be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention. The appendedclaims include all such embodiments and equivalent variations.

1. A non-naturally-occurring nucleic acid construct for expressing atleast one cistron in a crustacean cell, the construct comprising a viruspromoter from a virus that infects shrimp and an insect picornavirus(IPV) internal ribosome entry site (IRES), wherein the promoter and theIRES are operably linked with the at least one cistron for expression ofthe cistron in the cell, wherein the promoter is selected from the groupconsisting of P2 and P61 of infectious hypodermal and hematopoieticnecrosis virus(IHHNV).
 2. The construct of claim 1, wherein the IRES iscapable of infecting crustaceans from the phylum Arthropoda.
 3. Theconstruct of claim 1, wherein the IRES is the IRES of an 5′-untranslatedregion of the insect picornavirus.
 4. The construct of claim 1, whereinthe IRES is the IRES of an intergenic region of the insect picornavirushaving at least two ORFs.
 5. The construct of claim 1, wherein the IRESis a Taura syndrome virus (TSV) IRES.
 6. The construct of claim 5,wherein the IRES is the IRES of the 5′-untranslated region of TSV. 7.The construct of claim 1, wherein the IRES is an IRES of an IPV thatinfects shrimp.
 8. The construct of claim 1, wherein the constructfurther comprises an animal cell origin of replication site (ORS). 9.The construct of claim 8, wherein the animal is a crustacean.
 10. Theconstruct of claim 9, wherein the crustacean is a shrimp.
 11. Theconstruct of claim 10, wherein the shrimp is a Penaeus shrimp.
 12. Theconstruct of claim 1, wherein the cistron comprises a first open readingframe (ORF).
 13. The construct of claim 12, wherein the ORF is notinterrupted by an intron.
 14. The construct of claim 12, wherein the ORFencodes a detectable marker.
 15. The construct of claim 12, furthercomprising a second ORF operably associated with the IRES.
 16. Theconstruct of claim 15, wherein at least one of the first and second ORFsencodes a protein not normally expressed in a shrimp.
 17. A method ofexpressing a protein in a crustacean cell, the method comprisingintroducing into the cell a non-naturally-occurring nucleic acidconstruct, the construct comprising a viral promoter from a virus thatinfects a crustacean and an IPV IRES, wherein an ORF encoding theprotein is operably linked with the promoter and the IRES, and whereinthe promoter is selected from the group consisting of P2 and P61 ofinfectious hypodermal and hematopoietic necrosis virus (IHHNV); andculturing the cell for expression of the protein.
 18. The method ofclaim 17, wherein the protein is a eukaryotic protein.
 19. The method ofclaim 17, wherein the protein is a shrimp protein.
 20. The method ofclaim 17, wherein the crustacean is a shrimp.
 21. The method of claim20, wherein the shrimp is a Penaeus shrimp.
 22. The method of claim 17,wherein the construct is a plasmid.
 23. The method of claim 17, whereinthe construct is introduced into the cell using a virus vector.
 24. Themethod of claim 17, wherein the construct is introduced into the cellballistically.
 25. The method of claim 17, wherein the construct isintroduced into the cell by electroporation.
 26. The method of claim 17,wherein the protein is a protein that exerts a protective effect on thecell.
 27. The method of claim 17, wherein the protein is a protein thatexerts a protective effect on an animal that comprises the cell.
 28. Themethod of claim 17, wherein the protein is a protein that exerts atherapeutic effect on the cell.
 29. The method of claim 17, wherein theprotein is a protein that exerts a therapeutic effect on an animal thatcomprises the cell.
 30. The method of claim 17, wherein the protein is aprotein that exerts a regulatory effect on expression of an endogenousgene of the cell.
 31. A method of expressing a protein in a marineanimal cell, the method comprising introducing into the cell anon-naturally-occurring nucleic acid construct, the construct comprisinga viral promoter from a virus that infects shrimp and an insectpicornavirus IPV internal ribosome entry site IRES, wherein an ORFencoding the protein is operably linked with the promoter and the IRESfor expression of the protein in a marine animal cell, and wherein thepromoter is selected from the group consisting of P2 and P61 ofinfectious hypodermal and hematopoietic necrosis virus (IHHNV), andculturing the cell for expression of the protein.
 32. Anon-naturally-occurring nucleic acid construct for expressing a cistronin a crustacean cell, the construct comprising a virus promoter from avirus that infects crustacean and an insect picornavirus (IPV) internalribosome entry site (IRES), wherein at least the promoter is operablylinked to the cistron, wherein the IRES is from a virus known to infectthe crustacean, wherein the promoter is selected from the groupconsisting of P2 and P61 of infectious hypodermal and hematopoieticnecrosis virus(IHHNV).
 33. A non-naturally-occurring nucleic acidconstruct for expressing at least one cistron in a host cell, theconstruct comprising a virus promoter and an insect picornavirus (IPV)internal ribosome entry site (IRES), wherein the promoter and the IRESare operably linked with the cistron for expression of the cistron inthe host cell, wherein the promoter is selected from the groupconsisting of p2 and p61 of infectious hypodermal and hematopoieticnecrosis virus (IHHNV) and wherein the host cell is selected from agroup consisting of bacteria, yeast, insect, fish, shellfish andmollusk.
 34. The construct of claim 33, wherein the IRES is the IRES ofthe 5′-untranslated region of a cricket paralysis-like picornavirus. 35.The construct of claim 33, wherein the cistron comprises at least oneopen reading frame (ORF).
 36. The construct of claim 35, wherein the ORFencodes a protein not normally expressed in the host cell.
 37. Theconstruct of claim 36, wherein the protein is expressed in the host cellin vitro or in vivo.
 38. The construct of claim 36, wherein the proteinis antimicrobial or antiviral.
 39. A non-naturally-occurring nucleicacid construct for expressing at least one cistron in a host cell, theconstruct comprising a virus promoter and an insect picornavirus (IPV)internal ribosome entry site (IRES), wherein the promoter and the IRESare operably linked with the cistron for expression of the cistron inthe host cell, wherein the promoter is selected from the groupconsisting of p2 and p61 of infectious hypodermal and hematopoieticnecrosis virus (IHHNV).
 40. The construct of claim 39, wherein the hostcell is selected from a group consisting of bacteria cell, yeast cell,insect cell, fish cell, shellfish cell, and animal cell.